The long-term goal of this project is to gain an understanding on the structure/function relationship of the ATP synthase as it relates to mechanism of the ATP synthase. Recently, in a collaborative study, we were able to obtain a 3.2-3.3 A map of the yeast F1 ATPase. This major advance now allows us to ask and answer questions using the full tools of yeast genetics and x-ray crystallography. The studies in this proposal, in part, investigate the molecular impact of mutations in genes encoding subunits of the mitochondrial ATP synthase on the structure and function of the ATP synthase. In first aim of this study, the crystal structure analysis will be completed and improved to even further resolution. The second aim will use both site directed and random mutagenesis to investigate the structure/function relationship of the interactions of the gamma-subunit. The third aim will investigate the role of nucleotide binding on forming the active sites of the enzyme, F1F0 ATP synthase. This final aim represents the most ambitious aim of the 4, but also represents the single most important remaining goal needed in the understanding of the mechanism of the mitochondrial ATP synthase. These experiments will provide a further understanding on the structure function of the ATP synthase, but will also provide important information on the functional consequences of mutations in subunits of he ATP synthase. Ultimately, this project will provide critical information on the regulation of the ATP synthase and the importance of this regulation on the overall capabilities of the cell to provide enough energy as demanded by tissue and organism in both the normal and disease state.